Journal: bioRxiv

Article Title: Spiking GABAergic OPC tumor cells prolong survival in IDH1 mutant glioma

doi: 10.1101/2024.03.02.583026

Figure Lengend Snippet: (a) Heatmap showing subtype-specific GABAergic neuron markers as well as established OPC markers in Allen Brain Atlas and the Human Protein Atlas (HPA). White dotted box outlines OPCs. (b) Percentage of OPCs that express the GABA-OPC transcriptional signature in non-tumor brain atlases. (c) DotPlot showing the average expression of GABARs and GABA metabolism genes in human non-tumor OPCs and in GABA-OPC tumor cells. (d) Heatmap showing the expression GABARs and GABA metabolism genes in Patch-seq data. (e) IHC staining for GAD1 (red), IDH1R132H (green), OLIG2 (pink) and Hoechst (blue) in a human IDH1 mut tumor sample. (f) DotPlot showing the average expression of voltage-gated sodium channels (Na v s) and voltage-gated potassium channels (K v s) genes in human non-tumor OPCs and also in GABA-OPC tumor cells (g) Heatmap showing the Na v s and K v s genes in Patch-seq data. (h) IHC staining for Na V 1.1 (SCN1A; red), IDH1R132H (green), OLIG2 (pink) and Hoechst (blue) in a human IDH1 mut tumor sample.

Article Snippet: The following primary antibodies were used: mouse anti-IDH1R132H (1:50; Dianova, DIA-H09), rabbit anti-OLIG2 (1:100; R&D, AF4788), rabbit anti-GAD1 (1:200; Synaptic Systems, 198013), rabbit anti-SCN1A (Na V 1.1) Antibody (1:200; Alomone Labs, ASC-001).

Techniques: Expressing, Immunohistochemistry

Journal: Scientific reports

Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization.

doi: 10.1038/s41598-023-44093-8

Figure Lengend Snippet: Figure 1. IGSF3 expression in the developing mouse organs. (A–F) Sagittal sections of mouse embryo at E13.5 stained with an anti-IGSF3 antibody and visualized in brown color. (A) Overall IGSF3 protein expression in the embryo. (B–F) Higher magnification panels show expression (marked by arrows) in the forebrain (B), the hindbrain (C), the craniofacium (D), small intestine (E), and dorsal root ganglia (arrow) and spinal cord (arrowhead) (F). (G–O) Sagittal sections of mouse embryo at E17.5 stained with anti-IGSF3 antibody and visualized in brown color. (G) Overall expression in the embryo. (H–O) Higher magnification panels show expression (marked by arrows) in the cerebral cortex of the brain (H), the cerebellum (arrow) and the choroid plexus (arrow head) (I), optic nerve (J), teeth (K), whiskers (arrow) and the craniofacium (L), developing enteric ganglionic plexi of the small intestine, insert shows higher magnification of the boxed area (M), dorsal root ganglia (N), and the spinal cord (O). Scale bars: (A) 1000 µm; (B–F) 200 µm; (G) 2000 µm; (H–O) 100 µm.

Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY343 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes 14 Vol:. (1234567890) Scientific Reports | (2023) 13:17162 | https://doi.org/10.1038/s41598-023-44093-8 for 3–4 h using 0.3% Triton-X-100 in PBS.

Techniques: Expressing, Staining

Journal: Scientific reports

Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization.

doi: 10.1038/s41598-023-44093-8

Figure Lengend Snippet: Figure 2. Both IGSF3 mRNA and protein are expressed in the neural crest cells. (A–F) Single cell RNA sequencing analysis at E8.5 and E9.5 represented as t-SNE plots reveals the neural crest (NC) and neural tube (NT) cell clusters defined by Sox10 (B,E) and Sox2 (C,F) expression, respectively. The subclusters are assigned according to the annotations from the original analysis of the source data23. Igsf3 is expression is restricted to the neural crest cells at E8.5 (A). At E9.5, Igsf3 is expressed predominantly by the delaminating and migrating neural crest which is identified by Sox10 with some expression in the neural tube cells (F). (G,H) Serial sections of E9.5 embryo were stained using the anti-IGSF3 and anti-SOX10 antibodies (red color). IGSF3 expression (G) is detected in the Sox10-positive (H) neural crest cells as indicated by arrows pointing to the neural crest cells that are newly migrating out of the dorsal neural tube on both sides. Scale bars: (G,H) 50 µm.

Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY343 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes 14 Vol:. (1234567890) Scientific Reports | (2023) 13:17162 | https://doi.org/10.1038/s41598-023-44093-8 for 3–4 h using 0.3% Triton-X-100 in PBS.

Techniques: RNA Sequencing, Expressing, Staining

Journal: Scientific reports

Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization.

doi: 10.1038/s41598-023-44093-8

Figure Lengend Snippet: Figure 3. Single-cell RNAseq analysis shows that Igsf3 is the only IGSF member that is expressed by the neural crest cells. (A) Large panel in the left upper corner displays the t-SNE embedding showing the cell clusters of E8.5 Wnt1-Cre; R26Tomatoembryo. The image is adopted from the original analysis and annotations of the source data, of which our analysis is based on23. The early neural crest comprises of three spatially distinct clusters including a Hox-negative (–) corresponding to the anterior cranial neural crest, Hoxb2- positive ( +) corresponding to the mandibular level, and HoxD3-positive ( +) corresponding to the post-otic (including cardiac and vagal) streams of the neural crest23. Single cell RNA sequence analysis reveals the expression of different Igsf members in the distinct cell clusters defined respectively by Sox10 (NC cells) and Sox2 expression (NT cells). Igsf3 is the only family member expressed solely by the neural crest cells at E8.5. The other IGSF members are expressed about equally by both cell clusters or preferentially by the neural tube cell cluster. Expression of Igsf5 and Igsf23 was not detected at E8.5. (B) Large panel in the left upper corner displays the t-SNE embedding showing the cell clusters of mouse E9.5 Wnt1Cre/R26RTomato embryo and reflects the spatiotemporal properties of neural crest (NC) development, as adopted from the original analysis of the source data23. These neural crest cell clusters are classified into the following major subpopulations such as pre-EMT, delaminating and migrating neural crest, sensory neurons, autonomic neurons, and mesenchyme23. Single cell RNA sequence analysis at E9.5 represented as t-SNE plot reveals the expression of various IgSF members in different neural tube (NT) and neural crest (NC) cell clusters defined by Sox10 and Sox2 expression, respectively. Igsf3 is expressed in the NC and its derivative cell clusters also at this stage. Igsf8 and Igsf9 are predominantly expressed in the NT population, while Igsf10 and Igsf9b are detected about equally in the NC and NT cells, and Igsf11 shows a slight preferential expression in the NC cell cluster. (C) At E10.5, the choroid plexus and NC cells specifically express Igsf3, but other Igsf members are not co-expressed in these cells.

Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY343 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes 14 Vol:. (1234567890) Scientific Reports | (2023) 13:17162 | https://doi.org/10.1038/s41598-023-44093-8 for 3–4 h using 0.3% Triton-X-100 in PBS.

Techniques: Sequencing, Expressing

Journal: Scientific reports

Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization.

doi: 10.1038/s41598-023-44093-8

Figure Lengend Snippet: Figure 5. Loss of Igsf3 impairs neural crest cell migration. (A–J) E9.0 WT and KO vagal (collected at somite level 1–7) and trunk (collected at somite level 9–12) neural tube explants were cultured on fibronectin-coated cover slips for 48 h after which they were imaged. (A,B) Vagal neural crest explants isolated from WT (A) and KO (B) embryos. (C,D) Panels show the zoomed-in view of the boxed areas in (A) and (B). (E,F) Trunk neural crest explants isolated from WT (E) and KO (F) embryos. (G,H) Panels show the zoomed-in view to the boxed areas in (E) and (F). (I,J) Quantification of the neural crest cell migration. The area containing the migrating cells is marked with a green line, and the migration distance between the outer edges of the halo and the explant (black) was measured. The vagal neural crest cells of the KO explants migrated significantly less than the WT neural crest cells (I) while no difference was detected in the trunk neural crest cell migration between the WTs and KOs (J) (N = 4 for each genotype). (K,L) Representative images of the dorsal root ganglia in WT (K) and KO (L) pups at P12.5 (N = 3 for each genotype). Neurons are visualized by Tuj1-staining (green) and nuclei with DAPI (blue). (M) Quantification of Tuj1-positive neurons in the dorsal root ganglia showed no difference between the WT and KO samples. Scale bars: (A,B) and (E,F) 1000 µm, (K,L) 100 µm.

Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY343 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes 14 Vol:. (1234567890) Scientific Reports | (2023) 13:17162 | https://doi.org/10.1038/s41598-023-44093-8 for 3–4 h using 0.3% Triton-X-100 in PBS.

Techniques: Migration, Cell Culture, Isolation, Staining

Journal: Scientific reports

Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization.

doi: 10.1038/s41598-023-44093-8

Figure Lengend Snippet: Figure 6. Loss of Igsf3 results in impaired development of intestinal muscularis propria. (A,B) Small intestines collected from the P9 WT and KO pups (N = 1 for each genotype) and stained with hematoxylin and eosin (H&E). (C–F) To visualize the developing muscle layers of the small intestine the sections were stained with antibodies against αSMA (blue in (C,D), red in (E,F). (C,D) show the whole intestinal section while the panels (E,F) show the higher magnification images. (G,H) Higher magnification H&E staining from a representative sample of the small intestine from WT (G) and KO (H) P12.5 pups (N = 3 for each genotype). The arrows indicate the thickness of the muscularis externa (muscularis propria). (I) Quantification of the thickness of the developing enteric muscularis externa from P12.5 pups shows a significant reduction in the KO intestine as compared to samples from WT littermate controls. N = 3 for each genotype. Data was measured from two sections/intestine, four images/section, and 10 measurements points per image. Scale bars; (C,D) 100 µm, (G,H) 20 µm.

Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY343 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes 14 Vol:. (1234567890) Scientific Reports | (2023) 13:17162 | https://doi.org/10.1038/s41598-023-44093-8 for 3–4 h using 0.3% Triton-X-100 in PBS.

Techniques: Staining

Journal: Scientific reports

Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization.

doi: 10.1038/s41598-023-44093-8

Figure Lengend Snippet: Figure 7. Igsf3 KO results in downregulation of neuronal and smooth muscle markers and disorganized innervation of the small intestinal villi. (A) Confocal images of the WT P12.5 small intestinal villi after wholemount staining using antibodies against IGSF3 and NCAM1. IGSF3 (green) colocalizes with NCAM1 (red), a neuronal marker, in the neurons of the small intestinal villi (Merged). (B) Representative images of the intestinal villi from P12.5 WT (upper panels) and Igsf3 KO (lower panels) pups stained with antibodies against the neuron marker Tuj1 (green) and smooth muscle marker αSMA (red) on whole mount samples. (C,D) Representative images of the intestine from P12.5 WT (C) and Igsf3 KO (D) pups stained with antibodies against the E-cadherin (Ecad, green) and NCAM1 (red) on whole mount samples. Cell nuclei are visualized by Hoechst (blue). While no difference was seen in the E-cadherin expression between the WT and KO intestine, NCAM1 expression was substantially reduced in the KO compared to the WT. (E) Western blot analysis of P2.5 cerebrum extracts shows reduced expression of NCAM1 in KO samples also in the brain. Housekeeping protein GAPDH was used as a loading control. (F–K) Quantification from P12.5 intestinal immunostained images. (F) Expression of both Tuj1 and (G) αSMA was significantly reduced. (H) The αSMA:Tuj1 ratio was significantly increased in the KO villi as compared to WTs. (I) Counts of nerves per villus was significantly decreased in the KO villi as compared to the WT intestine, (J) but no difference was observed in the muscle fiber counts. (K) The colocalization of αSMA-Tuj1 was reduced in the KO villi as compared to WT intestines. Scale bars: (A–D) 100 µm.

Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY343 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes 14 Vol:. (1234567890) Scientific Reports | (2023) 13:17162 | https://doi.org/10.1038/s41598-023-44093-8 for 3–4 h using 0.3% Triton-X-100 in PBS.

Techniques: Staining, Marker, Expressing, Western Blot, Control

Journal: Scientific Reports

Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization

doi: 10.1038/s41598-023-44093-8

Figure Lengend Snippet: IGSF3 expression in the developing mouse organs. ( A – F ) Sagittal sections of mouse embryo at E13.5 stained with an anti-IGSF3 antibody and visualized in brown color. ( A ) Overall IGSF3 protein expression in the embryo. ( B – F ) Higher magnification panels show expression (marked by arrows) in the forebrain ( B ), the hindbrain ( C ), the craniofacium ( D ), small intestine ( E ), and dorsal root ganglia (arrow) and spinal cord (arrowhead) ( F ). ( G – O ) Sagittal sections of mouse embryo at E17.5 stained with anti-IGSF3 antibody and visualized in brown color. ( G ) Overall expression in the embryo. ( H – O ) Higher magnification panels show expression (marked by arrows) in the cerebral cortex of the brain ( H ), the cerebellum (arrow) and the choroid plexus (arrow head) ( I ), optic nerve ( J ), teeth ( K ), whiskers (arrow) and the craniofacium ( L ), developing enteric ganglionic plexi of the small intestine, insert shows higher magnification of the boxed area ( M ), dorsal root ganglia ( N ), and the spinal cord ( O ). Scale bars: ( A ) 1000 µm; ( B – F ) 200 µm; ( G ) 2000 µm; ( H – O ) 100 µm.

Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY3 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes for 3–4 h using 0.3% Triton-X-100 in PBS.

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization

doi: 10.1038/s41598-023-44093-8

Figure Lengend Snippet: Both IGSF3 mRNA and protein are expressed in the neural crest cells. ( A – F ) Single cell RNA sequencing analysis at E8.5 and E9.5 represented as t-SNE plots reveals the neural crest (NC) and neural tube (NT) cell clusters defined by Sox10 ( B , E ) and Sox2 ( C , F ) expression, respectively. The subclusters are assigned according to the annotations from the original analysis of the source data . Igsf3 is expression is restricted to the neural crest cells at E8.5 ( A ). At E9.5, Igsf3 is expressed predominantly by the delaminating and migrating neural crest which is identified by Sox10 with some expression in the neural tube cells ( F ). ( G , H ) Serial sections of E9.5 embryo were stained using the anti-IGSF3 and anti-SOX10 antibodies (red color). IGSF3 expression ( G ) is detected in the Sox10-positive ( H ) neural crest cells as indicated by arrows pointing to the neural crest cells that are newly migrating out of the dorsal neural tube on both sides. Scale bars: ( G , H ) 50 µm.

Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY3 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes for 3–4 h using 0.3% Triton-X-100 in PBS.

Techniques: RNA Sequencing Assay, Expressing, Staining

Journal: Scientific Reports

Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization

doi: 10.1038/s41598-023-44093-8

Figure Lengend Snippet: Single-cell RNAseq analysis shows that Igsf3 is the only IGSF member that is expressed by the neural crest cells. ( A ) Large panel in the left upper corner displays the t-SNE embedding showing the cell clusters of E8.5 Wnt1-Cre; R26 Tomato embryo. The image is adopted from the original analysis and annotations of the source data, of which our analysis is based on . The early neural crest comprises of three spatially distinct clusters including a Hox -negative (–) corresponding to the anterior cranial neural crest, Hoxb2 -positive ( +) corresponding to the mandibular level, and HoxD3 -positive ( +) corresponding to the post-otic (including cardiac and vagal) streams of the neural crest . Single cell RNA sequence analysis reveals the expression of different Igsf members in the distinct cell clusters defined respectively by Sox10 (NC cells) and Sox2 expression (NT cells). Igsf3 is the only family member expressed solely by the neural crest cells at E8.5. The other IGSF members are expressed about equally by both cell clusters or preferentially by the neural tube cell cluster. Expression of Igsf5 and Igsf23 was not detected at E8.5. ( B ) Large panel in the left upper corner displays the t-SNE embedding showing the cell clusters of mouse E9.5 Wnt1Cre/R26R Tomato embryo and reflects the spatiotemporal properties of neural crest (NC) development, as adopted from the original analysis of the source data . These neural crest cell clusters are classified into the following major subpopulations such as pre-EMT, delaminating and migrating neural crest, sensory neurons, autonomic neurons, and mesenchyme . Single cell RNA sequence analysis at E9.5 represented as t-SNE plot reveals the expression of various IgSF members in different neural tube (NT) and neural crest (NC) cell clusters defined by Sox10 and Sox2 expression, respectively. Igsf3 is expressed in the NC and its derivative cell clusters also at this stage. Igsf8 and Igsf9 are predominantly expressed in the NT population , while Igsf10 and Igsf9b are detected about equally in the NC and NT cells, and Igsf11 shows a slight preferential expression in the NC cell cluster. ( C ) At E10.5, the choroid plexus and NC cells specifically express Igsf3, but other Igsf members are not co-expressed in these cells.

Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY3 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes for 3–4 h using 0.3% Triton-X-100 in PBS.

Techniques: Sequencing, Expressing

Journal: Scientific Reports

Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization

doi: 10.1038/s41598-023-44093-8

Figure Lengend Snippet: Deletion in the exon 3 of Igsf3 gene leads near to complete loss of the IGSF3 protein. The CRISPR-Cas9 mediated genome engineering targeted exon 3 of the Igsf3 gene. ( A ) Schematic presentation of the Igsf3 inactivation strategy. Deleted nucleotides span across the sgRNA binding sites. The red star at the end of the nucleotide sequence indicates the pre-mature stop codon. ( B ) Gene analysis reveals a 146-nucleotide deletion in the KO allele, which resulted in a premature stop codon right after the signal peptide of Igsf3 . PAM protospacer adjacent motif. ( C , D ) The effect of Igsf3 deletion at the mRNA level was evaluated by using qPCR of the P2.5 cerebrum ( C ) and intestine ( D ). In heterozygous tissues, no significant differences in the Igsf3 levels were detected at the mRNA level whereas no Igsf3 transcript was detected in the KO tissue samples. ( E ) HEK293FT cells were transfected with plasmid encoding the murine Igsf3 gene. Cell extracts were prepared 48 h post-transfection and IGSF3 expression was analyzed using Western blot. WT and mock-transfected HEK293FT were used as controls. Arrow points to the band with correct molecular weight. (F ) IGSF3 gene deletion was validated at the protein level from P2.5 cerebrum extracts by using Western blot analysis. The KO showed a nearly complete loss of protein expression while reduced expression was detected in the heterozygous brain samples. ( G ) The IGSF3 protein expression of the cerebrum samples was quantified by normalizing it to the house keeping protein GAPDH. ( H – M ) The successful loss of the Igsf3 in the KO animals was further validated by immunostaining the myenteric plexus of the intestinal wall on whole mount samples from P12.5 mice using anti-NCAM1 ( H , K ) and anti-IGSF3 ( I , L ) antibodies (N = 3 for each genotype). No IGSF3 signal was detected in the KO intestines ( L , M ) while expression was clearly visible in the WT ( I , J ) and it colocalized with the NCAM1. Scale bars: ( G – L )100 µm. NCAM levels in ( H ) and ( K ) have been enhanced to visualize the neuronal network since NCAM1 expression is reduced in the KO compared to the WT.

Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY3 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes for 3–4 h using 0.3% Triton-X-100 in PBS.

Techniques: CRISPR, Binding Assay, Sequencing, Transfection, Plasmid Preparation, Expressing, Western Blot, Molecular Weight, Immunostaining

Journal: Scientific Reports

Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization

doi: 10.1038/s41598-023-44093-8

Figure Lengend Snippet: Loss of Igsf3 impairs neural crest cell migration. ( A – J ) E9.0 WT and KO vagal (collected at somite level 1–7) and trunk (collected at somite level 9–12) neural tube explants were cultured on fibronectin-coated cover slips for 48 h after which they were imaged. ( A , B ) Vagal neural crest explants isolated from WT ( A ) and KO ( B ) embryos. ( C , D ) Panels show the zoomed-in view of the boxed areas in ( A ) and ( B ). ( E , F ) Trunk neural crest explants isolated from WT ( E ) and KO ( F ) embryos. ( G , H ) Panels show the zoomed-in view to the boxed areas in ( E ) and ( F ). ( I , J ) Quantification of the neural crest cell migration. The area containing the migrating cells is marked with a green line, and the migration distance between the outer edges of the halo and the explant (black) was measured. The vagal neural crest cells of the KO explants migrated significantly less than the WT neural crest cells ( I ) while no difference was detected in the trunk neural crest cell migration between the WTs and KOs ( J ) (N = 4 for each genotype). ( K , L ) Representative images of the dorsal root ganglia in WT ( K ) and KO ( L ) pups at P12.5 (N = 3 for each genotype). Neurons are visualized by Tuj1-staining (green) and nuclei with DAPI (blue). ( M ) Quantification of Tuj1-positive neurons in the dorsal root ganglia showed no difference between the WT and KO samples. Scale bars: ( A , B ) and ( E , F ) 1000 µm, (K , L ) 100 µm.

Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY3 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes for 3–4 h using 0.3% Triton-X-100 in PBS.

Techniques: Migration, Cell Culture, Isolation, Staining

Journal: Scientific Reports

Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization

doi: 10.1038/s41598-023-44093-8

Figure Lengend Snippet: Loss of Igsf3 results in impaired development of intestinal muscularis propria. ( A , B ) Small intestines collected from the P9 WT and KO pups (N = 1 for each genotype) and stained with hematoxylin and eosin (H&E). ( C – F ) To visualize the developing muscle layers of the small intestine the sections were stained with antibodies against αSMA (blue in ( C , D ), red in ( E , F ). ( C , D) show the whole intestinal section while the panels ( E , F ) show the higher magnification images . ( G,H) Higher magnification H&E staining from a representative sample of the small intestine from WT ( G ) and KO ( H ) P12.5 pups (N = 3 for each genotype). The arrows indicate the thickness of the muscularis externa (muscularis propria). ( I ) Quantification of the thickness of the developing enteric muscularis externa from P12.5 pups shows a significant reduction in the KO intestine as compared to samples from WT littermate controls. N = 3 for each genotype. Data was measured from two sections/intestine, four images/section, and 10 measurements points per image. Scale bars; ( C , D ) 100 µm, ( G , H ) 20 µm.

Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY3 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes for 3–4 h using 0.3% Triton-X-100 in PBS.

Techniques: Staining

Journal: Scientific Reports

Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization

doi: 10.1038/s41598-023-44093-8

Figure Lengend Snippet: Igsf3 KO results in downregulation of neuronal and smooth muscle markers and disorganized innervation of the small intestinal villi. ( A ) Confocal images of the WT P12.5 small intestinal villi after wholemount staining using antibodies against IGSF3 and NCAM1. IGSF3 (green) colocalizes with NCAM1 (red), a neuronal marker, in the neurons of the small intestinal villi (Merged). ( B ) Representative images of the intestinal villi from P12.5 WT (upper panels) and Igsf3 KO (lower panels) pups stained with antibodies against the neuron marker Tuj1 (green) and smooth muscle marker αSMA (red) on whole mount samples. ( C , D ) Representative images of the intestine from P12.5 WT ( C ) and Igsf3 KO ( D ) pups stained with antibodies against the E-cadherin (Ecad, green) and NCAM1 (red) on whole mount samples. Cell nuclei are visualized by Hoechst (blue). While no difference was seen in the E-cadherin expression between the WT and KO intestine, NCAM1 expression was substantially reduced in the KO compared to the WT. ( E ) Western blot analysis of P2.5 cerebrum extracts shows reduced expression of NCAM1 in KO samples also in the brain. Housekeeping protein GAPDH was used as a loading control. ( F – K ) Quantification from P12.5 intestinal immunostained images. ( F ) Expression of both Tuj1 and ( G ) αSMA was significantly reduced. ( H ) The αSMA:Tuj1 ratio was significantly increased in the KO villi as compared to WTs. ( I ) Counts of nerves per villus was significantly decreased in the KO villi as compared to the WT intestine, ( J ) but no difference was observed in the muscle fiber counts. ( K ) The colocalization of αSMA-Tuj1 was reduced in the KO villi as compared to WT intestines. Scale bars: ( A – D ) 100 µm.

Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY3 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes for 3–4 h using 0.3% Triton-X-100 in PBS.

Techniques: Staining, Marker, Expressing, Western Blot

Journal: JCI Insight

Article Title: IGSF3 mutation identified in patient with severe COPD alters cell function and motility

doi: 10.1172/jci.insight.138101

Figure Lengend Snippet: (A and B) Complete karyotype in a lymphoblastoid cell line generated from patients’ lymphocytes showing a balanced translocation (B, arrows) between G-banded chromosomes 1 and 4: t(1;4)(p13.1;q34.3). der(1), derivative chromosome 1; der(4), derivative chromosome 4. (C–F) Metaphase FISH analysis of the translocation breakpoint. (C) BAC clones CTD-2329H4 and CTC-779C7on chromosome 1p13.1 show intact signals. (D) BAC clone RP4-686J16 on chromosome 1p13.1 shows green split signals between der(1) and der(4). (E) BAC clone RP11-763N18 on chromosome 4q34.3 shows the split signals of der(1) and der(4) (green), while those of RP11-495H13 (chromosome 4q34.3; orange) are intact. (F) DNA fiber FISH analysis demonstrating the translocation breakpoint within BAC clones RP4-686J16 (chromosome 1p13.1; green) and RP11-763N18 (chromosome 4q34.3; orange). (G) Schematic illustrating the translocation breakpoint regions on chromosomes 1p and 4q. Genomic locations shown are based on the UCSC genome browser (hg38). (H and I) Expression levels of IGSF3 mRNA measured by qPCR targeted array (n = 6 independent experiments, normalized to 18S, H) and of IGSF3 protein detected by immunoblotting (I, β-actin was loading control) in transformed lymphoblastoid cells derived from patient lymphocytes compared with lymphoblastoids derived from healthy donor lymphocytes (control). ****P < 0.0001 vs. control, Student’s t test.

Article Snippet: Sheep anti–human IGSF3 polyclonal antibody (1:25; R&D Systems, catalog AF4788) was biotinylated and added to the sections and incubated overnight at 4°C.

Techniques: Generated, Translocation Assay, Clone Assay, Expressing, Western Blot, Control, Transformation Assay, Derivative Assay

Journal: JCI Insight

Article Title: IGSF3 mutation identified in patient with severe COPD alters cell function and motility

doi: 10.1172/jci.insight.138101

Figure Lengend Snippet: (A) IGSF3 mRNA in lungs of C57BL/6 mice exposed to ambient air control (AC) or CS for 1 day. *P < 0.05 vs. AC, Student’s t test. (B) Representative images (from n = 5) of IGSF3 abundance detected by IHC in lungs of DBA2/J mice exposed to AC or CS for 4 months. Scale bar: 100μm. br, bronchus; v, vessel. (C) Quantification of IGSF3 abundance detected by IHC in lungs of DBA2/J mice exposed to AC or CS for 4 months. *P < 0.05 vs. AC, Student’s t test.

Article Snippet: Sheep anti–human IGSF3 polyclonal antibody (1:25; R&D Systems, catalog AF4788) was biotinylated and added to the sections and incubated overnight at 4°C.

Techniques: Control

Journal: JCI Insight

Article Title: IGSF3 mutation identified in patient with severe COPD alters cell function and motility

doi: 10.1172/jci.insight.138101

Figure Lengend Snippet: (A and B) Presence of IGSF3 and indicated proteins detected by immunoblots of density gradient fractions from lymphoblastoids (A) and bronchial epithelial cells (B). (C) IGSF3 and ITGB1 detected by Western blotting in lung epithelial cells after immunoprecipitation of density gradient fractions 9 and 12 with anti-CD9 or IgG control antibodies. (D) Presence of IGSF3 or indicated proteins in either streptavidin-bound or in flow through fractions of lung epithelial cells that were cell surface biotinylated with or without CS exposure. (E) Identification of IGSF3 protein and indicated protein markers of cellular organelles in density gradient fractions of lung endothelial cells. EEA1, early endosome antigen 1; PDI, protein disulfide-isomerase; LC3B, microtubule-associated protein 1 light chain 3 β; GM130, Golgi matrix protein 130 KDa; SDHA, succinate dehydrogenase complex flavoprotein subunit A; CytC, cytochrome c.

Article Snippet: Sheep anti–human IGSF3 polyclonal antibody (1:25; R&D Systems, catalog AF4788) was biotinylated and added to the sections and incubated overnight at 4°C.

Techniques: Western Blot, Immunoprecipitation, Control

Journal: JCI Insight

Article Title: IGSF3 mutation identified in patient with severe COPD alters cell function and motility

doi: 10.1172/jci.insight.138101

Figure Lengend Snippet: (A and B) Heatmap of expression levels of genes involved in sphingolipid (A) and ganglioside (B) metabolism relative to endogenous IGSF3 expression levels, measured by cDNA microarray in lymphoblastoids from patient and 5 healthy controls. (C–G) mRNA expression levels of indicated enzymes determined by targeted qPCR. Dots represent independent experiments; mean ± SEM shown as horizontal lines; *P < 0.05; ****P < 0.001, unpaired Student’s t test.

Article Snippet: Sheep anti–human IGSF3 polyclonal antibody (1:25; R&D Systems, catalog AF4788) was biotinylated and added to the sections and incubated overnight at 4°C.

Techniques: Expressing, Microarray

Journal: JCI Insight

Article Title: IGSF3 mutation identified in patient with severe COPD alters cell function and motility

doi: 10.1172/jci.insight.138101

Figure Lengend Snippet: (A and B) Caspase-3 activity in control and patient lymphoblastoid cells exposed to ultraviolet energy (UV; A) or to CS extract (CSE); n = 3–6 experiments, 1-way ANOVA. (C) Immunoblot of control and patient lymphoblastoids exposed to CSE (5% v/v) for the indicated time. (D) Heatmap of mRNA expression levels of genes involved in apoptosis, relative to IGSF3 expression. Red depicts higher relative expression, and blue represents lower relative expression. (E) Secreted proteins from patient lymphoblastoids (dark gray) compared with controls (light gray) exposed to CS; mean ± SEM; *P < 0.05; **P < 0.01; ***P< 0.001; 2-way ANOVA.

Article Snippet: Sheep anti–human IGSF3 polyclonal antibody (1:25; R&D Systems, catalog AF4788) was biotinylated and added to the sections and incubated overnight at 4°C.

Techniques: Activity Assay, Control, Western Blot, Expressing

Journal: JCI Insight

Article Title: IGSF3 mutation identified in patient with severe COPD alters cell function and motility

doi: 10.1172/jci.insight.138101

Figure Lengend Snippet: (A) Cell adhesion of patient lymphoblastoids to a monolayer of cultured lung epithelial cells (Beas2B, n = 3 with at least 2 replicates each, Student’s t test). (B) Cell migration of patient lymphoblastoids to a chemotactic stimulus (FBS) compared with controls (n = 5 with 3–6 replicates each, Student’s t test). (C) Immunoblot of IGSF3 in human bronchial epithelial cells Beas2B, stably transduced with control or IGSF3 shRNA. Actin was used as loading control. (D) Epithelial cell adhesion to fibronectin (1 hour; n = 4, P < 0.001, Student’s t test). (E) Immunoblot of IGSF3 in Beas2B transduced with control or IGSF3 CRISPR-CAS9. Vinculin was used as loading control. (F) Epithelial cell adhesion to fibronectin measured in Beas2B cells with CRISPR-CAS9–mediated knockdown of IGSF3 and expressed as fold change vs. control (n = 4 experiments, P < 0.05, Student’s t test).

Article Snippet: Sheep anti–human IGSF3 polyclonal antibody (1:25; R&D Systems, catalog AF4788) was biotinylated and added to the sections and incubated overnight at 4°C.

Techniques: Cell Culture, Migration, Western Blot, Stable Transfection, Transduction, Control, shRNA, CRISPR, Knockdown

Journal: JCI Insight

Article Title: IGSF3 mutation identified in patient with severe COPD alters cell function and motility

doi: 10.1172/jci.insight.138101

Figure Lengend Snippet: (A) IGSF3 immunoblot of Beas2B stably transfected with IGSF3 shRNA or pEF-IGSF3 with V5-HIS tag and respective controls. (B) Wound healing assay measured as closure (%) of a scratch wound in Beas2B cells stably expressing shRNA to IGSF3 (n = 9, ****P < 0.001, Student’s t test) or overexpressing pEF-IGSF3-V5His (n = 3, with 4 replicates, ****P < 0.001, Student’s t test). (C and D) IGSF3 immunoblot following knockdown in primary rat endothelial cells (RLEC) transfected with control or IGSF3 shRNA followed by closure (%) of a scratch wound (n = 6, ****P < 0.001). (E) Wound healing in primary HMVECL transfected with IGSF3 siRNA or control siRNA (n = 5, **P < 0.01, Student’s t test). (F) Epithelial cell barrier function measured by electric cell impedance sensing (ECIS) expressed as transcellular electrical resistance (TER) normalized to the TER at the time at which CS extract was added, in confluent Beas2B cell monolayers transfected with IGSF3 shRNA or control shRNA and exposed to CS (7.5%) or ambient air control (AC) extracts (n = 6–8, *P < 0.05, **P < 0.01, Student’s t test).

Article Snippet: Sheep anti–human IGSF3 polyclonal antibody (1:25; R&D Systems, catalog AF4788) was biotinylated and added to the sections and incubated overnight at 4°C.

Techniques: Western Blot, Stable Transfection, Transfection, shRNA, Wound Healing Assay, Expressing, Knockdown, Control